l-706000 and clofilium

Block of human ether-a-go-go related gene (HERG) K(+) channels by a variety of medications has been linked to acquired long QT syndrome, a disorder of cardiac repolarization that predisposes to lethal arrhythmias. The drug-binding site is composed of residues that face into the central cavity of the channel. Two aromatic residues located on the S6 domain (Tyr652 and Phe656) are particularly important structural determinants of drug block. The role of pore helix residues (Thr623, Ser624, Val625) is less clear. In this study, we compared the pharmacological properties of two structurally related compounds, ibutilide and clofilium. Both compounds are charged amines with a single phenyl ring. Clofilium, a chlorobenzene derivative, is a potent blocker of HERG channels, but has a remarkably slower time course for recovery from block than ibutilide, a methanesulfonanilide. The difference in the rate of recovery from block can be explained simply by variation in drug trapping. There is little recovery from clofilium block with D540K HERG channels that permit untrapping at hyperpolarized potentials. Alanine-scanning mutagenesis of the S6 domain and a portion of the pore helix revealed that the binding site residues were the same for both compounds. However, S624A, located at the base of the pore helix, was the only HERG mutation that enabled rapid recovery from clofilium block. In summary, the pore helix residues are important components of the HERG drug binding site, and may be particularly important for drugs with polar substituents, such as a halogen (e.g., clofilium) or a methanesulfonamide (e.g., ibutilide).

Topics: Amino Acid Substitution; Animals; Anti-Arrhythmia Agents; Aspartic Acid; Benzopyrans; Binding Sites; ERG1 Potassium Channel; Ether-A-Go-Go Potassium Channels; Humans; Kinetics; Models, Molecular; Mutagenesis, Site-Directed; Oocytes; Phenethylamines; Phenylalanine; Piperidines; Potassium Channel Blockers; Potassium Channels; Potassium Channels, Voltage-Gated; Pyridines; Quaternary Ammonium Compounds; Serine; Sulfonamides; Tyrosine; Xenopus laevis

This investigation determined the effects of K(+) channel antagonists on proliferation, differentiation, and apoptosis of porcine granulosa cells. The drugs screened for functional effects included the class III antiarrhythmic agents MK-499 and clofilium, the chromanol I(Ks) antagonist 293B, the benzodiazepine I(Ks) antagonists L-735,821 and L-768,673, and the peptidyl toxins charybdotoxin (CTX) and margatoxin (MTX). Granulosa cell proliferation and differentiation were assessed by serial measurements of cell number and progesterone accumulation in the culture media, respectively. Granulosa cell apoptosis was evaluated using flow cytometry. Additional information about drug effects was obtained by immunoblotting to detect expression of proliferating cell nuclear antigen, p27(kip1) and the caspase-3 substrate poly(ADP-ribose) polymerase. The ERG channel antagonist MK-499 had no functional effects on cultured granulosa cells. However, the broad spectrum K(+) channel antagonist clofilium decreased, in a concentration-dependent fashion, the number of viable granulosa cells cultured, and these effects were associated with induction of apoptosis. All three I(Ks) antagonists (293B, L-735,821, and L-768,673) increased basal, but not FSH-enhanced progesterone accumulation on Day 1 after treatment without affecting the number of viable cells in culture, an effect that was blocked by pimozide. In contrast, CTX and MTX increased the number of viable cells in FSH-stimulated cultures on Day 3 after treatment without affecting progesterone output per cell. These data demonstrate that selective antagonism of granulosa cell K(+) channels with distinct molecular correlates, electrophysiological properties, and expression patterns can influence differential granulosa cell proliferation, steroidogenic capability, and apoptosis. Thus, K(+) channels may represent pharmacological targets for affecting Granulosa cell function and oocyte maturation, in vivo or in vitro.

Topics: Animals; Apoptosis; Benzopyrans; Blotting, Western; Caspase 3; Caspases; Cell Count; Cell Differentiation; Cell Division; Cells, Cultured; Charybdotoxin; Female; Flow Cytometry; Granulosa Cells; Membrane Potentials; Neurotoxins; Piperidines; Potassium Channel Blockers; Potassium Channels; Quaternary Ammonium Compounds; Scorpion Venoms; Steroids; Swine